Structure–Activity Relationships Reveal Beneficial Selectivity Profiles of Inhibitors Targeting Acetylcholinesterase of Disease-Transmitting Mosquitoes

Insecticide resistance jeopardizes the prevention of infectious diseases such as malaria and dengue fever by vector control of disease-transmitting mosquitoes. Effective new insecticidal compounds with minimal adverse effects on humans and the environment are therefore urgently needed. Here, we explore noncovalent inhibitors of the well-validated insecticidal target acetylcholinesterase (AChE) based on a 4-thiazolidinone scaffold. The 4-thiazolidinones inhibit AChE1 from the mosquitoes Anopheles gambiae and Aedes aegypti at low micromolar concentrations. Their selectivity depends primarily on the substitution pattern of the phenyl ring; halogen substituents have complex effects. The compounds also feature a pendant aliphatic amine that was important for activity; little variation of this group is tolerated. Molecular docking studies suggested that the tight selectivity profiles of these compounds are due to competition between two binding sites. Three 4-thiazolidinones tested for in vivo insecticidal activity had similar effects on disease-transmitting mosquitoes despite a 10-fold difference in their in vitro activity.


■ INTRODUCTION
Vector-borne diseases caused by viruses, parasites, and bacteria cause health, social, and economic problems that affect large numbers of people worldwide, especially in tropical and subtropical low-and middle-income countries. Mosquitoes are key vectors that transmit pathogens to the humans and animals on which they feed. In particular, mosquitoes of the Anopheles, Aedes, and Culex genera spread diseases such as malaria, dengue, chikungunya, and West Nile fever. In this study, we focus on the mosquito species Aedes aegypti (Ae. aegypti) and Anopheles gambiae (An. gambiae) that transmit, for example, dengue fever and malaria, respectively. Malaria alone was linked to over 600 000 deaths in 2020 according to the latest report by the World Health Organization (WHO). 1 Although the estimated numbers of malaria infections and related deaths have decreased significantly since 2000, the incidence of infection has plateaued over the past seven years and increased in 2019 due to the disruption of services caused by the COVID-19 pandemic. 1, 2 The most efficient intervention for suppressing the spread of mosquito-borne diseases is vector control, which relies heavily on the effectiveness of the active ingredients in adulticides and larvicides. For example, insecticide-treated nets and indoor residual spraying played major roles in reducing the number of clinical cases of malaria between 2000 and 2015. 2 However, the extensive use of a small number of insecticide classes has led to growing insecticide resistance in mosquito populations, threatening the effectiveness of current vector control strategies. 3−8 Consequently, there is an urgent need to develop new active ingredients with different mechanisms of action. Additional tools for vector control that are being studied include biocontrol, 9−12 genetic engineering of mosquitos, 9,13 new chemical tools such as semiochemicals 14 and nanoparticles, 15 and strategies using various combinations of these tools. 16 An important molecular target for mosquito control is the enzyme acetylcholinesterase (AChE), which is present in both humans and animals. 17,18 This essential enzyme terminates cholinergic neurotransmission by rapidly degrading the neurotransmitter acetylcholine in the synaptic cleft. Structural studies have shown that AChE has a deep and narrow active site gorge that is lined with aromatic amino acid residues. 19 Three loops at the entrance of the gorge constitute the peripheral site (loops 1 and 2, plus the Ω loop), while the active site features a catalytic triad (serine, histidine, and glutamate) and is located near the bottom of the gorge ( Figure 1A). Although humans and mammals express one AChE enzyme, most insects including mosquitoes express two AChE isoforms, designated AChE1 and AChE2. 20 AChE1 is the more strongly expressed isoform in mosquitoes 21 and is known to hydrolyze the same substrates as its vertebrate analogues, including human AChE (hAChE), with similar kinetic properties. 22,23 Structural and sequence comparisons of An. gambiae AChE1 (AgAChE1) and hAChE1 have shown that their active sites are identical but there are some differences in the three loops at the entrance of the gorge ( Figure  1B). The conservation of the AChE active site across diverse organisms is an important disadvantage for two of the most widely used AChE-targeting insecticide classes, organophosphates and carbamates, because they both inhibit AChE by covalent bonding with the serine of the catalytic triad. 18,24 This can cause severe poisoning and death in humans who are overexposed to these insecticides while applying them, and also gives rise to off-target ecotoxicity in beneficial species such as honeybees. Furthermore, mutations that confer resistance to these agents in disease-transmitting mosquitoes by preventing covalent inhibition occur in the active site gorge of AChE1. One of the most widespread resistance-conferring mutations is G122S-AgAChE1, 25 which is not present in Ae. aegypti expressing AeAChE1.
To overcome the problems of toxicity towards other species and growing insecticide resistance, several attempts have been made to develop new chemical entities with the same mechanism of action as organophosphates and carbamates. 26−34 Although improvements have been achieved, it has been difficult to obtain both selectivity for AChE1 over hAChE and potency against the resistance-conferring mutant G122S-Ag-AChE1. [27][28][29]31,33 An approach based on a different mechanism of action involves designing covalent inhibitors targeting a cysteine residue near the active site of AgAChE1 (Cys447 Ag ), which is mutated into Phe295 h in hAChE ( Figure 1B), in order to increase selectivity. 35,36 While this approach is interesting, recent findings have raised questions about its viability because the compounds developed to date lack appreciable selectivity, and are proposed to have a different mode of irreversible binding not related to cysteine-targeted binding. 37 Furthermore, no mosquitocidal activity has been reported for cysteine-targeting inhibitors. As an alternative to targeting the active site, there have been efforts to develop noncovalent inhibitors, which could potentially complement covalent inhibitors. 38−42 Although studies in this area have yielded promising results, more research is needed to fully evaluate the potential of such inhibitors as active ingredients for vector control. Interestingly, phenotypic screening of compound libraries 43,44 and plant extracts 45,46 against mosquito larvae have revealed compounds targeting AChE1, highlighting its importance as a molecular target for a wider range of chemicals than organophosphates and carbamates. To clarify the potential of noncovalent inhibitors to serve as safe and efficient active ingredients, we here explore the inhibitory activity of a new chemical scaffold against AChE1 enzymes from the mosquitoes An. gambiae and Ae. aegypti. Our investigation focuses on the structure−activity relationships (SARs) for potency against wild-type AChE1 and selectivity over hAChE as well as identifying prerequisites for G122S-AgAChE1 inhibition.

Identification of 4-Thiazolidinones as AgAChE1
Inhibitors. In a previously reported high-throughput screening (HTS) of 17 000 compounds against recombinantly expressed AgAChE1 and AeAChE1, 23,42 we identified 4-thiazolidinones 4 and 18 as potential inhibitors of mosquito AChE1 (Figure 2). (B) AgAChE1 (pink ribbons) superpositioned to hAChE (gray ribbons) and amino acid residues important for inhibitor binding are displayed as sticks. Loop 1 is colored light and dark blue, α-helix/loop 2 is colored yellow and orange, and Ω loop is colored light and dark green for AgAChE1 and hAChE, respectively. The figures are based on protein coordinates of previously published crystal structures (PDB codes 5X61 and 4EY4, for AgAChE1 and hAchE, respectively).
Compounds of this class are discussed extensively in the medicinal chemistry and patent literature because they exhibit diverse biological activities including anticancer, antibiotic, antiviral, antifungal, and cardiovascular activity. Consequently, their potential applications have been reviewed in detail. 47,48 It has also been suggested that 4-thiazolidinones could be used to treat insect-borne diseases such as malaria 49,50 and dengue fever. 51 However, their use as insecticides for vector control has not previously been investigated to our knowledge. The most closely related compounds used for this purpose appear to be rhodanine derivatives 52,53 bearing a thiocarbonyl in the 2position of the ring, whose electronic properties and reactivity differ significantly from our HTS hits ( Figure 2).
Several publications have described the synthesis of 4thiazolidinones from commercially available reagents. [48][49][50][51]54 The 4-thiazolidinone scaffold is readily prepared via a multicomponent reaction between an aldehyde (1), thioglycolic acid (2), and an amine (3) in which the starting materials are refluxed together using a Dean−Stark trap or molecular sieves to remove water (Scheme 1). We adapted this approach to prepare 4thiazolidinones starting from diamines 3 and used microwaveassisted synthesis to shorten reaction times while achieving yields comparable to those of refluxing methods. The microwave-assisted strategy permits the use of diverse functional groups in starting materials 1 and 3, allowing a wide range of compounds to be prepared in a simple one-pot process. We also designed a stepwise synthesis of these compounds for use in cases where the desired amine 3 is not commercially available (Scheme 1, bottom). In this alternative pathway, the initial multicomponent 4-thiazolidinone synthesis is performed using an amine with a terminal alcohol group, which is then transformed into the corresponding bromide via the Appel reaction, allowing the desired product to be finally obtained via an S N 2 reaction. Using the one-pot approach, our screening hits 4 and 18 were synthesized as racemic mixtures in one step. Moreover, after performing an extractive work-up to remove water-soluble impurities, 4 and 18 were conveniently isolated as easily-handled solid salts by slow addition of HCl in ether to induce precipitation followed by filtration. This allowed us to verify the activity of 4 and 18 against AgAChE1; their halfmaximal inhibitory concentrations (IC 50 ) were determined to be 5.4 and 0.86 μM, respectively, confirming the activity observed in the HTS. In addition, both 4 and 18 showed weak activity against hAChE, with IC 50 values above 200 μM.
Design Strategy. We designed two sets of 4-thiazolidinones for synthesis and biochemical evaluation. The first set had 4 as their parent compound (Design 1a), while the second set was derived from 18 (Design 1b; Scheme 2). The set 1a compounds were designed to investigate the effects of varying the substituents of the aliphatic amine and of replacing the amine itself with hydroxy, azido, aromatic, or heteroaromatic groups (Table S1). The effect of varying the length of the pendant carbon chain was also investigated. The set 1b compounds were designed to investigate the effect of modifying the aromatic ring by adding electron-donating or electron-withdrawing substituents at various positions, introducing fused rings, or replacing the phenyl ring with a heteroarene (Table 1). We then designed a third set of derivatives (Design 2) that combined the aromatic substituents and alkyl chain lengths with the most promising effects on activity from designs 1a and 1b while retaining the terminal dimethylamine moiety of 4 and 18 (Tables 2 and S2). Finally, a fourth set (Design 3; Scheme 2) was designed in which Scheme 1. One-Pot and Stepwise Synthesis of 4-Thiazolidinone Analogues a a the best aromatic scaffolds from design sets 1 and 2 were combined with various secondary amine motifs (Tables 3 and  S3). All of the designed and synthesized compounds were tested for potency towards AgAChE1 and hAChE by measuring their IC 50 values. Some compounds were also tested against the mutant G122S-AgAChE1 and AaAChE1, and a few were selected for enzyme kinetics studies and evaluation of their in vivo activity against An. gambiae and Ae. aegypti.
Design 1a: Exploration of N-Substitution Based on 4. All compounds in set 1a (5−17, Table S1) were synthesized as shown in Scheme 1 and tested for inhibitory activity against AgAChE1 and hAChE. If the 2-phenyl-4-thiazolidinone unit was left unmodified, modifying the pendant amine moiety by changing the N-substituents had a clear adverse effect on AgAChE1 inhibition; no compounds modified in this way exhibited superior or even similar potency to 4. None of the analogues of 4 achieved complete inhibition of the enzyme even at high concentrations (1 mM). Shortening the chain length as in 5 reduced inhibitory potency toward the mosquito enzyme considerably compared to 4 (IC 50 = 161 vs 5.4 μM, respectively). Replacing the dimethylamino moiety with a diethylamino group also reduced activity in analogues with 3-or 2-carbon alkyl chains (6 and 7, respectively). Similar results were observed upon replacing the dimethylamino unit with a heterocyclic substituent such as a morpholine (8), imidazole (14,15), or triazole (16). Compounds lacking a pendant amino group (9,10,17) showed no detectable inhibitory activity towards AgAChE1 even at a concentration of 1 mM. The reference compound 4 and all the analogues shown in Table S1 exhibited little activity against hAChE even at high concentrations. Overall, these experiments clearly showed that retaining the pendant dimethylamine moiety with a 3-carbon chain is important for AgAChE1 inhibition.
Design 1b: Exploration of Aromatic Substitution Based on 18. The compounds in set 1b, in which the aromatic moiety of lead compound 18 was modified, are listed in Table 1 and were synthesized following Scheme 1. Biochemical evaluation of these compounds revealed that the types and positions (ortho, meta, or para) of the substituents on the phenyl ring strongly affected AgAChE1 inhibition. For example, shifting the para chlorine atom of the parent compound 18 to the ortho position (as in compound 20) or the meta position (as in compound 19) increased the IC 50 values from 0.86 to 8.1 μM and 16 μM, respectively. However, the ortho methoxysubstituted compound 23 had an IC 50 value of 2.5 μM against AgAChE1, whereas its para and meta substituted analogues 21 and 22 were less active (IC 50 = 53 and 85 μM, respectively). The para compound 24 was the strongest inhibitor of the three fluorinated analogues (0.82 μM), followed by the meta substituted 25 and the ortho substituted 26 (IC 50 = 14 and 23 μM, respectively). Replacing the phenyl ring with a pyridine moiety reduced activity against the mosquito enzyme; the pyridinyl compounds 27, 28, and 29 were the weakest inhibitors of this subset. The set 1b compounds were substantially less active against hAChE than AgAChE1; only the meta chlorinated compound 19 exhibited even moderate inhibition of hAChE (IC 50 = 25 μM). The other analogues were even less active toward the human enzyme ( Table 2). For example, the para and ortho chlorinated analogues 18 and 20 had IC 50  Overall, the para substituted chlorinated and fluorinated analogues 18 and 24 were the strongest inhibitors. Encouragingly, these two were also poor inhibitors of hAChE and thus showed the highest selectivity in the subset. The least selective compound was the meta chlorinated compound 19, which was equipotent against the human and mosquito enzymes.
Design 2: Follow-Up Design Based on Initial Findings. In Design 2, we investigated the effect of varying the aromatic moiety in more detail by examining disubstituted analogues bearing Cl and F atoms in different positions ( Table 2). We also examined the effect of replacing the phenyl ring with larger aromatic moieties such as naphthyl and benzodioxole groups. All of the 11 analogues in this set had a pendant 3-carbon alkyl chain with a terminal dimethylamino moiety like the parent compound 18 and the compounds listed in Table 1. Separately, we prepared some analogues bearing the new aromatic groups with a 2-carbon dimethylamino chain to see if the loss of activity due to modification of the pendant amine would be as noticeable as in set 1a (Table S2).
The evaluation of these compounds against AgAChE1 (Table  2) showed that the naphthyl analogue 36 had the lowest IC 50 value of the series (0.65 μM), meaning that larger substituents were tolerated. However, the increased hydrophobic character of 36 reduced its solubility at higher concentrations. The ortho, para dichlorinated compound 30 had a similar inhibitory Scheme 2. Design Strategy Based on the Two Hit Compounds potency to its para monosubstituted counterpart. On the other hand, the ortho, para difluorinated analogue 31 had an IC 50 of 4.4 μM, while compound 32, which contains both chlorine and fluorine, had an IC 50 of 3.0 μM. The ortho, meta dichlorinated compound 34 (3.6 μM) was a weaker inhibitor of AgAChE1 than its ortho, para dichlorinated analogue 30 but was a stronger binder than the corresponding difluoro species 33 (37 μM). Replacing the phenyl group with a benzodioxole moiety, as in 35, reduced inhibitory activity towards AgAChE1. The twocarbon chain analogues 37, 38, 39, and 40 were substantially weaker than their 3-carbon chain counterparts, confirming the results obtained with set 1a (Tables S1 and S2). In addition, the biochemical evaluations indicated that the SAR of these compounds for inhibition of hAChE differed from that for inhibition of AgAChE1. Based on their IC 50 values, most of the tested compounds (including 30, 31, and 32) were at least 20 times weaker inhibitors against human AChE than against the mosquito enzyme, and 36 was 2 orders of magnitude stronger binder against the mosquito enzyme. Interestingly, the compounds with ortho, meta disubustituted aromatic moieties (33 and 34) were rather strong inhibitors against the human enzyme, with IC 50 values of 7.2, and 4.4 μM, respectively. As a result, 34 was an equipotent inhibitor of the human and mosquito enzymes, while 33 inhibited the human enzyme even more strongly than AgAChE1. The 2-carbon chain compounds  (Table S2).

Design 3: Focused Exploration of Secondary Amines.
In the third design set, we synthesized and biochemically evaluated compounds to investigate the effect of replacing the  pendant tertiary amine with a secondary amine. We combined three of the most promising aromatic scaffolds (24, 30, and 36) with four secondary amines connected to the 4-thiazolidinone core by 2-and 3-carbon chains: an isopropylamine, a cyclopropylamine, and a monomethylamine connected via a 2carbon chain, and a monomethylamine connected via a 3-carbon chain (Tables 3 and S3). Four compounds combining the 2carbon chain isopropylamine moiety with fluorinated aromatic scaffolds (cf. 55, 56, 57, and 58) were also investigated; see Table S3.
The compounds with more sterically demanding secondary amines (cyclopropylamines and isopropylamines) were stronger inhibitors of AgAChE1 than the compounds with monomethyl amines. The most active compound in this series was 46, which has an ortho, para dichlorophenyl ring and a pendant cyclopropylamine; its IC 50 value was 2.3 μM. The second most active compound was its isopropyl analogue 41 (4.2 μM), while the methylamine analogues 50 and 52 were less active (Table 3). Interestingly, the ortho and para chlorinated analogues were stronger inhibitors than their para fluorinated and naphthyl counterparts when the secondary amine was introduced (Tables 3 and S3) even though these aromatic substituents conferred similar activity in the previous compound sets featuring pendant tertiary amines. Among the para fluorophenyl compounds, the cyclopropylamine 49 had the lowest IC 50 value (11 μM), followed by the isopropylamine analogue 42 (18 μM). The naphthyl compounds 54 and 43 were over 10 times weaker binders than their tertiary amine analogues. The other fluorinated scaffolds gave IC 50 values above 30 μM when combined with a pendant isopropylamine. The ortho, para dichlorophenyl compound with a pendant methylamine unit (50) exhibited the highest activity against hAChE in this set, with an IC 50 value of 49 μM; 50 was the only compound in this set that completely inhibited the human enzyme in vitro (Tables 3 and S3). Biochemical Evaluation against the Insecticide-Resistant Mutant Enzyme G122S-AgAChE1. Some of the synthesized 4-thiazolidinones were selected for evaluation against the insecticide-resistant mutant G122S-AgAChE1 (Tables 1−3, S2 and S3) based on their activity profiles against the wild-type mosquito and human enzymes. The results showed that the 4-thiazolidinones also inhibited the enzymatic activity of G122S-AgAChE1, albeit with lower potency and a different SAR to that observed for the wild-type enzyme. The most active compounds had an ortho chlorinated aromatic ring, and a tertiary amine group: the singly substituted ortho chloro compound 20 had an IC 50 value of 53 μM against G122S-AgAChE1, whereas the ortho chloro, para fluoro compound 32 and the ortho, meta dichloro compound 34 had IC 50 values of 63 and 62 μM, respectively. The ortho, para dichloro compound 30 was slightly less active against the mutant enzyme (117 μM). The para chloro lead compound 18 was substantially less active against the mutant; its IC 50 value could not be determined under our assay conditions, indicating that it was above 200 μM, compared to 0.86 μM for wild-type AgAChE1. The mutation of a glycine to a serine at position 122 in the binding gorge thus profoundly alters the 4-thiazolidinone SAR for the mosquito enzymes. This was further confirmed by the inactivity of the naphthyl analogue 36 against G122S-AgAChE1 (IC 50 > 200 μM) despite its good activity against wild-type AgAChE1 (IC 50 = 0.65 μM).
Structure−Activity Relationships of 4-Thiazolidinones Inhibiting AChE. Variation of the aromatic substituent of the 4-thiazolidinone scaffold revealed that chlorinated and fluorinated inhibitors were the most promising inhibitors of AgAChE, with the para substituted inhibitors 18 and 24 being particularly strong binders. Interestingly, the compounds' inhibition profiles for the studied AChEs depended on their substitution patterns: the para substituted compounds 18 and 24 showed the highest selectivity for the wild-type mosquito enzyme over the human enzyme but exhibited poor activity against the resistant mutant G122S-AgAChE (Figure 3a,b). Conversely, the ortho chloro analogue 20 exhibited greater potency against G122S-AgAChE but was a weaker inhibitor of AgAChE, leading to poor selectivity over hAChE (Figure 3c). Its fluorinated analogue 26 was a poor binder overall, and therefore less attractive ( Figure  3d). Substitution at both the ortho and para positions in the dichlorinated 30 and the difluorinated 31 led to increased inhibitory activity against both AgAChE and hAChE (Figure 3e−f), but 30 was a weaker inhibitor of the resistant mutant than its ortho monochlorinated counterpart 20. Changing the disubstitution pattern from ortho/para to ortho/meta (34 and 33) dramatically altered the inhibition profiles: both the dichloro and difluoro ortho, meta analogues were completely unselective for the mosquito enzyme, and the ortho, meta difluoro compound 33 was actually a stronger inhibitor against the human enzyme than against AgAChE (Figure 3g,h). Removing the halogen from the ortho position to obtain the singly-halogenated meta substituted compounds 19 and 25 led to a general loss of inhibitory activity in the case of 19, but the meta fluorinated compound 25 was a stronger inhibitor than 33 against AgAChE while being weaker against hAChE and thus regained selectivity for the mosquito enzyme (Table 1).
An SAR analysis of the pendant dimethylamine using analogues of the para fluorinated compound 24 and the ortho/para dichlorinated compound 30 revealed that the monofluorinated scaffold was more sensitive to modification of the pendant alkylamine (Tables 3 and S3). For example, replacing the 3-carbon chain dimethylamine moiety with a cyclopropylamine connected via a 2-carbon chain led to a 13fold loss of potency for the para fluorinated scaffold (49 vs 24) but only a 2-fold loss of potency for the ortho, para dichlorinated scaffold (46 vs 30). A similar pattern was seen for the isopropylamines (cf. 42 and 41 vs 24 and 30, respectively). The inhibition profiles of the cyclopropylamine derivatives closely resembled those of the 3-carbon chain dimethylamines.
Molecular Docking of 4-Thiazolidinones to AChEs. To further investigate the SAR of 4-thiazolidinones, we performed molecular docking simulations of eight inhibitors to AgAChE1, hAChE, and the mutant enzyme G122S-AgAChE1. The chosen inhibitors had fluoro or chloro substituents in the ortho and/or para positions of the phenyl ring together with a pendant 3carbon dimethylamine chain ( Figure S5). The three-dimensional (3D) structures of the enzymes used in this analysis were previously determined by X-ray crystallography (PDB codes 5X61, 4EY4/6O5V and 6ARX for AgAChE1, hAChE, and G122S-AgAChE1, respectively). The crystal structures of AChEs reveal several waters in the active site gorge, thus we have performed docking to AChE structures with included and excluded water molecules (Tables S23−S27). The R and S enantiomers of the inhibitors with positively charged dimethylamines were separately docked into the binding gorge of the AChEs using the Schrodinger software package and multiple docking poses for each inhibitor were analyzed.
The docking studies clearly showed that the inhibitors adopted different binding poses in each of the target enzymes. The dockings with included waters gave in general fewer poses. In AgAChE1 without waters, the docked 4-thiazolidinones had two main poses: pose A, in which they bound at the bottom of the gorge, close to the catalytic triad; and pose B, in which they bound at the peripheral site ( Figure 4). The dockings to AgAChE1 structure with waters resulted in fewer number of poses of type A, although still observed. Only pose B was observed when docking with hAChE without waters. Interestingly, when waters were included it revealed that in order for inhibitors to form pose B, a water molecule had to move ( Figure  5). Docking with G122S-AgAChE1 with and without waters generally yielded few promising poses, most of which had significantly lower docking scores than those for the other two enzymes. Nevertheless, pose B was also observed for G122S-AgAChE1. The enantiomeric preferences of each binding pose were complex. For AgAChE1, pose A was only observed for the S enantiomers, whereas pose B could be adopted by both R and S enantiomers in AgAChE1 (with and without waters) and hAChE (without waters). The few promising type B poses of 4thiazolidinones binding to G122S-AgAChE1 all involved R enantiomers.
In AgAChE1, the 4-thiazolidinones bind to the active site (pose A) via two main interactions: an interaction between the charged protonated nitrogen of the dimethylamine moiety and Trp245 Ag (cf. Trp86 h ), and an arene−arene stacking interaction between the phenyl rings of Tyr493 Ag (cf. Tyr341 h ) and the 4thiazolidinone. The aromatic rings of Tyr489 Ag and Phe490 Ag also participate in many-body arene−arene interactions with the 4-thiazolidinones, as previously reported for inhibitors of mouse AChE (cf. Tyr337 h and Phe338 h ). 55,56 A superposition of the structures of AgAChE and hAChE showed that the side chains of Tyr489 Ag /Tyr337 h have different conformations in these enzymes. The conformation of Tyr489 Ag makes the binding pocket of AgAChE somewhat larger than that of hAChE, which allowed the 4-thiazolidinones to be docked into the active site of the former but not the latter. This is consistent with the findings of Carlier et al., whose studies on carbamate AChE1 inhibitors revealed that larger βand γ-branched 1-alkylpyrazol-4-yl methylcarbamates are more selective for inhibition of AgAChE versus hAChE than their smaller analogues. 33 The G122S mutation is not close in space to the docked pose A (the shortest heavy atom distance between the mutant residue and the inhibitor is 5.2 Å), so there does not appear to be a direct steric clash that would prevent the 4-thiazolidinones from adopting binding poses at the bottom of the gorge. The binding gorge residue exhibiting the largest conformational difference between G122S-AgAChE and AgAChE is Tyr493 Ag (cf. Tyr341 h ), which makes the gorge in the G122S mutant slightly narrower than that in the wild-type enzyme. This may explain the lack of A-type binding poses for the mutant.
Both enantiomers of all of the 4-thiazolidinones binding in pose B formed similar interactions at the peripheral sites of the studied AChE species: the phenyl moiety formed a non-optimal arene−arene interaction with Trp441 Ag /Trp286 h ; the carbonyl of the thiazolidinone ring formed a hydrogen bond with backbone NH moieties of amino acid residues in loop 1; and the 3-carbon chain with the positively charged (protonated) dimethylamine moiety projected down into the gorge and formed N + CH−arene interactions with Tyr493 Ag /Tyr341 h , Tyr489 Ag /Tyr337 h , or Phe490 Ag /Phe338 h . In AgAChE1, the interacting moieties of the R and S enantiomers overlapped whereas in hAChE there were more pronounced differences between the enantiomers: the S enantiomers formed hydrogen bonds with the NH of Phe295 h , whereas the R enantiomers formed hydrogen bonds with Arg296 h . Both B poses in hAChE resulted in clashes between the −CH 2 − part of the fivemembered ring, and the water molecule forming H-bonds with Ser293 h and Arg296 h (Figure 5a). None of the investigated crystal structures (Tables S24 and S25) showed ligands replacing this water molecule. The differences between the binding poses and the water patterns in AgACHE and hAChE depended on amino acid sequence differences in loop 1 and the Ω-loop. Loop 1 of AgAChE1 is three residues shorter than the corresponding loop in hAChE, and the Ile231 Ag residue in the Ω-loop of the mosquito enzyme is replaced by a tyrosine residue (Tyr72 h ) in the human enzyme, which explained the conformational differences between the corresponding docked poses and differences in water interactions. For G122S-AgAChE, only the R enantiomers adopted pose B and the binding pose of the 4thiazolidinones was similar to that seen with the wild-type enzyme. However, the N + CH−arene interactions differed because the conformation of Tyr493 Ag in G122S-AgAChE differs from that in the human and wild-type mosquito enzymes (as discussed above), which probably explains why the docking scores for the mutant were lower.
Comparing the docking poses of the different compounds more closely, the docking studies suggested that the ortho and para substituted 4-thiazolidinones were able to bind in a deeper position within the binding gorge of AgAChE (pose A) than in hACHE and G122S-AgAChE, which is consistent with their selectivity profiles as inhibitors. The −Cl and −F substituents of 18 and 24 formed contacts with hydrogens of the aromatic ring of Phe490 Ag (cf. Phe338 h ) and the β carbon of Tyr493 Ag (cf. Tyr341 h ), respectively, suggesting that electrostatic interactions involving these halogen substituents may favor this binding mode. In contrast, pose B at the peripheral site could be adopted by all docked compounds in all three investigated AChE species, although a water molecule needed to be replaced in hAChE. It should be noted that the side chain of Trp286 h can adopt different conformations in mammalian AChE in the presence of different ligands, as demonstrated by X-ray crystallography. 57 Few crystal structures of AgAChE have been published, so there is insufficient experimental data to draw conclusions about the mobility of amino acid side chains in the peripheral site of this enzyme. However, based on amino acid sequence differences we can assume that it differs from that in hAChE. We therefore hypothesize that the observed dependence of inhibitory potency on the substitution pattern of the phenyl ring in 4-thiazolidinone derivatives is partly due to interactions with Trp441 Ag /Trp286 h in the binding site. Accordingly, we propose that the greater potency of the ortho, meta disubstituted compounds 33 and 34 towards hAChE and their reduced selectivity compared to other inhibitors are due to favorable interactions with Trp286 h in conformations other than the docked poses. In all of the binding poses, the pendant dimethylamine moiety formed key interactions with aromatic enzyme residues, in accordance with the SAR analysis.
Evaluation of Insecticidal Effects of 4-Thiazolidinones on Disease-Transmitting Mosquitoes. The insecticidal potential of selected 4-thiazolidinones was evaluated against the mosquito species An. gambiae and Ae. aegypti. First, 23 compounds were tested for inhibitory activity against recombinantly expressed AaAChE1, which confirmed that their IC 50 values for both mosquito enzymes were comparable and that they exhibited similar SAR patterns ( Figure 6, Tables 1−3, S2 and S3). Three 4-thiazolidinones with different potency (20, 24, and 30) were then selected for further investigation. Inhibition kinetics was used to determine the compounds' inhibition constant (K i ) and mode of inhibition ( Table 4). The selected compounds were investigated for inhibition of AgAChE1, G122S-AgAChE1, and hAChE using different substrate and inhibitor concentrations (Tables S28−S34). We found that the inhibitors decreased the maximum velocity (V max ) in a dose-dependent manner. In contrast, the K m values (substrate concentration at 50% of V max ) were constant. These observations are consistent with a noncompetitive inhibition mechanism for all compounds and enzymes. The kinetic inhibition constants of 20, 24, and 30 determined by noncompetitive inhibition curve fitting of kinetic data are shown in Tables 4 and S35. For AgAChE1, the kinetic inhibition constants were in good agreement with the IC 50 values, while there were some differences for G122S-AgAChE1 and hAChE. Compound 30, with a poor IC 50 value of 117 μM, was shown to have a moderate K i value of 19 μM toward G122S-AgAChE1. In contrast, inhibition of hAChE was slightly weaker according to the K i values of 20 and 30, compared to the IC 50 measurements.
Topical application of 20, 24, and 30 to An. gambiae and Ae. aegypti resulted in efficiently killed mosquitoes, causing 100% mortality in both species at a dosage of 3 μg per mosquito (10 nmol/mosquito). The compounds also showed a clear dose− response pattern: insecticidal activity decreased markedly (to approximately 50% mortality) when the dosage was reduced to 0.3 μg per mosquito, and was not detectable when the dosage was reduced further to 0.03 μg (Figure 7). It is interesting that the 4-thiazolidinones were equally effective against both An. gambiae and Ae. aegypti because previous studies have found that Ae. aegypti is less sensitive to AChE1 inhibitors than Ag. gambiae. 41,42,58 The estimated LD 50 value of 0.3 μg/mosquito for the 4-thiazolidinones is approximately 55 and 170 times higher, respectively, than the LD 50 values of the currently used insecticides propoxur (LD 50 = 0.0054 μg/mosquito) and bendiocarb (LD 50 = 0.0018 μg/mosquito), 58 but 7 times lower than the those of the previously reported noncovalent phenoxyacetamide-based inhibitors. 42 Discrepancies between in vitro and in vivo potency for compounds containing aliphatic amine moieties have previously been attributed to poor penetration of the exoskeleton. 42 However, the agreement between the in vivo and in vitro results for the 4-thiazolidinones was better than that for previously reported insecticidal compounds containing aliphatic amines such as thiourea-and phenoxyacetamide-based AChE1 inhibitors 41,42 and dopamine Figure 6. In vitro inhibition of AChE1 enzymes from mosquito species An. gambiae (x-axis) and Ae. aegypti (y-axis) by the studied 4thiazolidinones expressed as pIC 50 . The square of the Pearson correlation coefficient r 2 for the two mosquito enzymes' pIC 50 values is 0.96. receptor antagonists. 59 Interestingly, the in vivo insecticidal efficacies of the three compounds did not differ greatly despite the pronounced differences in their in vitro potency. A similar lack of correlation between AChE1 inhibition in vitro and mosquito LD 50 values was previously observed for carbamates. 33 These findings illustrate the complexity of testing with whole organisms and the importance of physicochemical and pharmacokinetic parameters whose influence is not reflected in the results of enzyme kinetics experiments.

■ CONCLUSIONS
The 4-thiazolidinones identified through HTS proved to be inhibitors of wild-type AChE1 enzymes from the mosquito species An. gambiae and Ae. aegypti. The in vitro SARs of these compounds were somewhat restrictive, however, because their inhibitory activity was highly sensitive to even minor modifications of their chemical structures. For example, any change in the structure of the pendant aliphatic tertiary amine moiety reduced inhibitory potency. Studies on compounds having differently substituted phenyl rings showed that the substitution patterns giving the strongest AChE1 inhibition differed from those giving the strongest hAChE inhibition, which is promising for avoiding off-organism toxic effects due to unwanted inhibition of human AChE. The problem of increasing insecticide resistance prompted us to test the most promising 4-thiazolidinones against the mutant G122S-Ag-AChE1 enzyme. This revealed that 4-thiazolidinones can inhibit the mutated AgAChE1, albeit with reduced potency and a different SAR from that seen for the wild-type enzyme. Thus, the challenge to obtain both high selectivity for AChE1 over hAChE and strong potency against the resistance-conferring mutant G122S-AgAChE1 needs to be pursued further. Molecular docking studies revealed some possible binding modes of these compounds and provided tentative structural explanations for the observed SAR trends. Notably, the 4-thiazolidinones docked into the lower part of the binding gorge of AgAChE1, which was not the case for hAChE and G122S-AgAChE1. In vivo experiments showed that the 4-thiazolidinones killed An. gambiae and Ae. aegypti mosquitoes. However, relatively high doses were needed and compounds with rather different potencies in vitro had similar insecticidal effects in vivo. This highlights the importance of considering factors in addition to in vitro potency against AChE1 in future efforts to develop novel noncovalent insecticidal agents. It also shows that within a given insecticidal efficacy window there may be several compounds with different selectivity and physicochemical profiles to choose from.
■ EXPERIMENTAL SECTION General Chemistry. All commercially available reagents and solvents (except dichloromethane) were purchased from Sigma-Aldrich, Acros, Fluorochem, Fisher Scientific, or VWR and used without further purification. Dichloromethane and DMF were dried in a solvent drying system (Glass Contour Solvent Systems, SG Water). 4 Å molecular sieves were activated at 180°C in the oven for at least 24 h prior to use. Microwave reactions were performed in sealed glass vials in Biotage Initiator EXP EU and Biotage Initiator+ EU (Biotage Sweden, AB) systems. When necessary, reaction conversion rates were monitored by TLC on aluminum sheets coated with silica gel 60 F 254 from Merck; spots were visualized by UV detection (254 nm) or by staining with KMnO 4 solution. Liquid chromatography−mass spectrometry (LC−MS) analyses were performed using a 6130 Quadrupole (Agilent Technologies) mass spectrometer connected to an Agilent 1260 Infinity LC system with an Agilent Proshell 120 EC-C18 2.7 μm 3 × 50 mm column. The eluent was H 2 O/CH 3 CN (0.1% HCOOH) and detection was performed at 210 and 254 nm. When necessary, compounds were purified with a Biotage Isolera One automated flash chromatography system (eluents given in brackets) using KP-SIL 50 μm or Biotage SNAP Ultra 25 μm silica gel disposable cartridges. NMR spectra were acquired on a Bruker DRX 400 or 600 MHz instrument at 298 K unless otherwise stated (Supporting Information, pages S42−S116). The δ values were referenced to the residual solvent signals of CDCl 3 (7.26 ppm), DMSO-d 6 (2.50 ppm), or CD 3 OD (3.31 ppm) as internal standards for 1 H and CDCl 3 (77.16 ppm), DMSO-d 6 (39.52 ppm), or CD 3 OD (49.00 ppm) as internal standards for 13 C. For 19 F NMR internal standard of CFCl 3 was used. All compounds are >95% pure by high-performance liquid chromatography (HPLC) analysis.
One-Pot Synthesis of Thiazolidinone Scaffold. Method A. The corresponding aldehyde (2 mmol) and the corresponding diamine (2 mmol) were added to a 100 mL round-bottom flask containing dry toluene (20 mL) and equipped with a magnetic bar, a reflux condenser, and 4 Å molecular sieves. The mixture was refluxed for 4 h, then allowed to cool down, then mercaptoacetic acid (6 mmol) was added while stirring, and the mixture was refluxed for 3 extra hours, and then allowed to cool down to 25°C. The organic solution was washed three times with an aqueous saturated NaHCO 3 solution (20 mL each), dried over Na 2 SO 4 , filtered, and solvent was removed under vacuum to afford a crude oil. Purification proceeded as detailed individually for each compound.
Method B. 60 In a microwave-suitable glass vial equipped with a magnetic bar and previously activated 4 Å molecular sieves, the corresponding amine 3 (2.0 mmol) was solved in absolute ethanol (99%, 4.5 mL) and the corresponding aldehyde 1 (4.0 mmol) and mercaptoacetic acid 2 (6.0 mmol) were added, the vial sealed with a suitable cap, and the reaction mixture heated up to 120°C by microwave irradiation and stirred at this temperature for 30 min. Then, the reaction mixture was allowed to cool down, remaining pressure excess released with a needle, and the reaction mixture was diluted with 50 mL of EtOAc, washed with 2 M NaOH aqueous solution (2 × 10 mL), distilled water (10 mL), and brine (20 mL). The organic phase

Journal of Medicinal Chemistry pubs.acs.org/jmc
Article was collected, dried over Na 2 SO 4 , and concentrated under vacuum to afford an oily crude containing unreacted aldehyde and the desired 4thiazolidinone. Purification proceeded as detailed individually for each compound. Method C. 60 In a microwave-suitable glass vial equipped with a magnetic bar and previously activated 4 Å molecular sieves, the corresponding amine 3 (2.0 mmol) was solved in absolute ethanol (99%, 4.5 mL) and the corresponding aldehyde 1 (4.0 mmol), mercaptoacetic acid 2 (6.0 mmol), and diisopropylethylamine (2.0 mmol) were added, the vial sealed with a suitable cap, and the reaction mixture heated up to 120°C by microwave irradiation and stirred at this temperature for 30 min. Then, the reaction mixture was allowed to cool down, remaining pressure excess released with a needle, and the reaction mixture was diluted with 50 mL of EtOAc, washed with 2 M NaOH aqueous solution (2 × 10 mL), distilled water (10 mL), and brine (20 mL). The organic phase was collected, dried over Na 2 SO 4 , and concentrated under vacuum to afford an oily crude containing unreacted aldehyde and the desired 4-thiazolidinone. Purification proceeded as detailed individually for each compound.
Conversion of Alcohols into Bromines. Method D. Following a modified experimental from the literature. 61 The corresponding alcohol (1.5 mmol) was dissolved in 5 mL of CH 2 Cl 2 and cooled down to 0°C. Triphenylphosphine (1.1 equiv) was added, followed by the addition of CBr 4 (1.1 equiv), and the mixture was stirred at 0°C. After 2 h of reaction time, TLC indicated full conversion. Then, the reaction mixture was concentrated in vacuo and the residue was purified by automated flash chromatography to afford the corresponding bromine. The bromine was used immediately for the next reaction due to suspected decomposition. (11). Synthesized following the Method A section in a 10 mmol scale. Crude purified by automated flash column chromatography using KP-Sil cartridge and an eluent gradient of Heptane/EtOAc (50:50 to 0:100) to afford 1.4 g of a colorless viscous oil (63% yield). Characterization data in accordance with previously reported in the literature. 62 3-(3-Bromopropyl)-2-phenylthiazolidin-4-one (12).    (14). NaH (0.40 mmol) was stirred in 4 mL of dry deaminated DMF, and the mixture cooled down to 0°C. Then, 1H-imidazole (0.40 mmol) was added, and the mixture was stirred at 0°C for 15 min. Then, a solution of the corresponding alkyl bromide (0.31 mmol) in dry deaminated DMF (1 mL) was added dropwise. The mixture was allowed to warm up to room temperature and stirred overnight. After, the reaction was diluted with EtOAc (15 mL) and washed 3 times with NaHCO 3 (aq sat., 5 mL each). The organic phase was extracted three times with HCl (aq, 1 M, 5 mL each). The acidic aqueous phase was made basic by the addition of NaOH pellets and extracted three times with EtOAc (5 mL each). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under vacuum to afford 31 mg of a yellow oil (35% yield).

3-(2-(1H-Imidazol-1-yl)ethyl)-2-phenylthiazolidin-4-one
Hydrochloride (15). NaH (0.40 mmol) was stirred in 4 mL of dry deaminated DMF, and the mixture cooled down to 0°C. Then, 1H-imidazole (0.40 mmol) was added, and the mixture was stirred at 0°C for 15 min. Then, a solution of the corresponding alkyl bromide (0.31 mmol) in dry deaminated DMF (1 mL) was added dropwise. The mixture was allowed to warm up to room temperature and stirred overnight. After, the reaction was diluted with EtOAc (15 mL) and washed 3 times with NaHCO 3 (aq sat., 5 mL each). The organic phase was extracted three times with HCl (aq, 1 M, 5 mL each). The acidic aqueous phase was made basic by the addition of NaOH pellets and extracted three times with EtOAc (5 mL each). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under vacuum. Purified by dissolution of the crude in 1 mL of ethyl acetate and precipitation by the dropwise addition of HCl in Et 2 O (1 M, 0.5 mL) while stirring. The solid was filtered to afford the product as white crystals (24 mg, 25% yield). 1 H NMR (600 MHz, DMSO-d 6  3-(2- (1H-1,2,4-triazol-1-yl)ethyl)-2-phenylthiazolidin-4-one (16). NaH (0.40 mmol) was stirred in 4 mL of dry deaminated DMF, and the mixture cooled down to 0°C. Then, 1,2,4-triazole (0.40 mmol) was added, and the mixture was stirred at 0°C for 15 min. Then, a solution of the corresponding alkyl bromide (0.31 mmol) in dry deaminated DMF (1 mL) was added dropwise. The mixture was allowed to warm up to room temperature and stirred overnight. After, the reaction was diluted with EtOAc (15 mL) and washed 3 times with NaHCO 3 (aq sat., 5 mL each). The organic phase was extracted three times with HCl (aq, 1 M, 5 mL each). The acidic aqueous phase was made basic by the addition of NaOH pellets and extracted 3 times with EtOAc (5 mL each). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under vacuum. Purified by trituration in Et 2 O to afford a pale-yellow solid ( (17). The corresponding alkyl bromide (0.33 mmol) was dissolved in a screw-capped vial with 3 mL of dry deaminated DMF. Then, NaN 3 (0.50 mmol) and potassium iodide (0.03 mmol) were added. The vial was sealed, and the mixture was heated to 80°C in an oil bath for 18 h while stirring. The mixture was poured into 15 mL of Et 2 O and washed three times with distilled water (5 mL each). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under vacuum to afford 72 mg of a yellow oil without further purification (82% yield). 1 (19). Synthesized following the Method A section. Purified by dissolution of the crude in 10 mL of ethyl acetate and  (20). Synthesized following the Method A section.  (26). Synthesized following a modified the Method A section. Once the reaction was complete, the compound was extracted with a solution of aqueous NaHCO 3 (3 times, 10 mL each). Then, the aqueous phase was extracted with CH 2 Cl 2 (3 times, 20 mL each). The organic phases were combined, dried over Na 2 SO 4 , filtered, and concentrated under vacuum to afford 304 mg of a yellow oil. No further purification was needed (57% yield). 1 (28). Synthesized following a modified the Method A section. Once the reaction was complete, the compound was extracted with a solution of aqueous NaHCO 3 (3 times, 10 mL each). Then, the aqueous phase was extracted with CH 2 Cl 2 (3 times, 20 mL each). The organic phases were combined, dried over Na 2 SO 4 , filtered, and concentrated under vacuum to afford 302 mg of a yellow oil. No further purification was needed (57% yield). 1 H NMR (400 MHz, Chloroform-d)  13 (31).  (32 (42). Synthesized following the Method C section. Purified by dissolution of the crude in 1 mL of methanol and 20 mL of diethyl ether, followed by precipitation by the dropwise addition of HCl in Et 2 O (1 M, 2 mL) while stirring. A yellowish goo was formed instead of crystals. Solvent was removed by pipette and the remaining material was triturated and sonicated with chloroform to afford 415 mg of paleyellow powder (65% yield). HPLC purity 99.4% 3-(2-(Isopropylamino)ethyl)-2-(naphthalen-1-yl)thiazolidin-4one Hydrochloride (43). Synthesized following the Method C section. Purified by dissolution of the crude in 1 mL of ethyl acetate and 20 mL of diethyl ether, followed by precipitation by the dropwise addition of HCl in Et 2 O (1 M, 2 mL) while stirring. The solid was filtered, washed with diethyl ether, and collected to afford 532 mg of pale-yellow crystals GraphPad Prism and the log [inhibitor] vs response variable slope equation was fitted using four parameters. For all four targets, the compounds were tested at least twice at different time points and with newly prepared dilutions from solid material of each replicate. Enzyme kinetics, including K i determinations, were made using a similar protocol as for the IC 50 experiments as described above. Eight concentrations of the substrate acetylthiocholine iodide were used based on the K m value for each enzyme 23 and kinetic experiments were run for each of the eight concentrations of the inhibitor, which were designed based on the IC 50 values. When preliminary data showed that the K i value differed from the IC 50 value, the concentrations were altered to improve the experimental design. The enzyme kinetic experiments were made in triplicates. Michaelis−Menten and noncompetitive inhibition curve fitting (nonlinear regression) were made in GraphPad Prism.

2-(4-Fluorophenyl)-3-(2-(isopropylamino)ethyl)thiazolidin-4one Hydrochloride
Molecular Docking. Dockings were made to crystal structures of AChE without and with included water molecules. Water molecules in the active site gorge were investigated using crystal structures with resolution ≤2.3 Å (Tables S23−S25). For the mosquito enzymes, only two G122S-AgAChE1 structures matched the criteria, while 20 hAChE and 19 mAChE structures were used for the human enzyme. The structures were superimposed and water molecules that were present in more than 60% of the structures were included in the dockings with waters (Tables S26 and S27). Water molecules that according to the crystal structures could be replaced by inhibitors were not included. The 3D coordinates of hAChE, AgAChE1, and G122S-AgAChE1 were obtained from previously published crystal structures (PDB codes 4EY4 (excluded waters), 6O5V (included waters), 5X61, and 6ARX), 64−66 and the structures were aligned with that of a previously reported noncovalent inhibitor mAChE complex (PDB code 5FUM). 42 The residue Tyr489 Ag (Tyr337 h ) was manually modified for each enzyme to match the "open" conformation of the residue in 5FUM. The structures were minimized with Macromodel using constraints on all heavy atoms with a constant force of 100 and a freedom value of 0.2 Å. The protein structures were then minimized and optimized with the Protein Preparation tool in Maestro, and the grid receptor was generated with Glide by using the aligned ligand from 5FUM as centroid, resulting in a cubic box of 20 Å length. Inhibitors were docked using Glide within the Schrodinger software 67 with Standard Precision settings, writing 50 poses per ligand and performing a postminimization on the resulting conformations. Finally, the inhibitors were ranked by docking score and docking poses with Glide score values ≤ −8.2 were visually analyzed.
Mosquito Rearing and In Vivo Testing with Anopheles and Aedes mosquitoes. The An. gambiae s.s. Kisumu and Ae. aegypti Mombasa strains, both from Kenya, were used for in vivo testing of the intrinsic activity of selected compounds. These mosquitoes have been colonized at KEMRI for over 20 years and are routinely tested to verify their susceptibility to permethrin and deltamethrin in accordance with WHO tube bioassay guidelines using diagnostic concentrations of 0.75% permethrin and 0.05% deltamethrin impregnated on filter paper. Mosquito rearing is conducted in an insectary maintained at 27−28°C and approximately 80% humidity on a 12/12 h light and darkness cycle and maintained at optimal larval concentrations to avoid possible effects of competition. Mosquito larvae are fed on finely ground Sera Vipan staple diet (Sera, Germany) and reared in tap water that is dechlorinated by allowing it to stand in a bucket in the insectary chamber for 24 h. Adult mosquitoes are fed on a fresh 10% (w/v) glucose solution meal daily and on hamster (Mesocricetus auratus) as a source of blood meals when egg production is desired.
In vivo testing of the compounds' intrinsic insecticidal activity was performed in accordance with WHO guidelines for testing adulticides and larvicides. This involves topical application of the compounds at different concentrations in acetone on the upper part of the protonum of mosquitoes using a micropipette. Five-day-old female non-blood-fed mosquitoes were used, and testing was performed on batches of 5 mosquitoes at a time. Each batch of 5 mosquitoes was placed in a 500 mL paper cup and anesthetized by placing the cup in a −20°C freezer for 3 min. Mosquitoes were then gently poured onto a freezer pack cooled to −20°C and overlaid with paper towel. The compound in acetone was then deposited on the upper part of the protonum. A total volume of 0.1 μL of the compound in acetone at the required concentration was applied to each mosquito. As a negative control, 0.1 μL of pure acetone was applied on some mosquitoes. After the topical application, the mosquitoes were returned to the paper cups and placed back in the insectary, where they were given a glucose meal and maintained under standard conditions. Mosquito mortality was recorded after 24